1. Field of the Invention
The present invention relates to enzyme-based biosensors for detecting noble gases.
2. Description of the Background
The ability of the noble gases helium (He), neon (Ne), argon (Ar), krypton (Kr), xenon (Xe) and radon (Ra) to enter into chemical combination with other atoms is extremely limited. Generally, only krypton, xenon and radon have been induced to react with other atoms, which are highly reactive, such as fluorine and oxygen, and the compounds thus formed are explosively unstable. See Advanced Inorganic Chemistry, by F. A. Cotton and G. Wilkinson (Wiley, Third Edition). However, while the noble gases are, in general, chemically inert, xenon is known to exhibit certain physiological effects, such as anesthesia. Other physiological effects have also been observed with other inert gases such as nitrogen, which, for example, is known to cause narcosis when used under great pressure in deep-sea diving.
It has been reported in U.S. Pat. No. 3,183,171 to Schreiner that argon and other inert gases can influence the growth rate of fungi and argon is known to improve the preservation of fish or seafood. U.S. Pat. No. 4,946,326 to Schvester, JP 52105232, JP 80002271 and JP 77027699. However, the fundamental lack of understanding of these observations clearly renders such results difficult, if not impossible, to interpret. Moreover, the meaning of such observations is further obscured by the fact that mixtures of many gases, including oxygen, were used in these studies. Further, some of these studies were conducted at hyperbaric pressures and at freezing temperatures. At such high pressures, it is likely that the observed results were caused by pressure damage to cellular components and to the enzymes themselves.
For example, from 1964 to 1966, Schreiner documented the physiological effects of inert gases particularly as related to anesthetic effects and in studies relating to the development of suitable containment atmospheres for deep-sea diving, submarines and spacecraft. The results of this study are summarized in three reports, each entitled: "Technical Report. The Physiological Effects of Argon, Helium and the Rare Gases," prepared for the Office of Naval Research, Department of the Navy. Contract Nonr 4115(00), NR: 102-597. Three later summaries and abstracts of this study were published.
One abstract, "Inert Gas Interactions and Effects on Enzymatically Active Proteins," Fed. Proc. 26:650 (1967), restates the observation that the noble and other inert gases produce physiological effects at elevated partial pressures in intact animals (narcosis) and in microbial and mammalian cell systems (growth inhibition).
A second abstract, "A Possible Molecular Mechanism for the Biological Activity of Chemically Inert Gases," In: Intern. Congr. Physiol. Sci., 23rd, Tokyo, restates the observation that the inert gases exhibit biological activity at various levels of cellular organization at high pressures.
Also, a summary of the general biological effects of the noble gases was published by Schreiner in which the principal results of his earlier research are restated. "General Biological Effects of the Helium-Xenon Series of Elements," Fed Proc. 27:872-878 ( 1968 ).
However, in 1969, Behnke et al refuted the major conclusions of Schreiner. Behnke et al concluded that the effects reported earlier by Schreiner are irreproducible and result solely from hydrostatic pressure, i.e., that no effects of noble gases upon enzymes are demonstrable. "Enzyme-Catalyzed Reactions as Influenced by Inert Gases at High Pressures." J. Food Sci. 34: 370-375.
In essence, the studies of Schreiner were based upon the hypothesis that chemically inert gases compete with oxygen molecules for cellular sites and that oxygen displacement depends upon the ratio of oxygen to inert gas concentrations. This hypothesis was never demonstrated as the greatest observed effects (only inhibitory effects were observed) were observed with nitrous oxide and found to be independent of oxygen partial pressure. Moreover, the inhibition observed was only 1.9% inhibition per atmosphere of added nitrous oxide.
In order to refute the earlier work of Schreiner, Behnke et al independently tested the effect of high hydrostatic pressures upon enzymes, and attempted to reproduce the results obtained by Schreiner. Behnke et al found that increasing gas pressure of nitrogen or argon beyond that necessary to observe a slight inhibition of chymotrypsin, invertase and tyrosinase caused no further increase in inhibition, in direct contrast to the finding of Schreiner.
The findings of Behnke et al can be explained by simple initial hydrostatic inhibition, which is released upon stabilization of pressure. Clearly, the findings cannot be explained by the chemical-O.sub.2 /inert gas interdependence as proposed by Schreiner. Behnke et al concluded that high pressure inert gases inhibit tyrosinase in non-fluid (i.e., gelatin) systems by decreasing oxygen availability, rather than by physically altering the enzyme. This conclusion is in direct contrast to the findings of Schreiner.
In addition to the refutation by Behnke et al, the results reported by Schreiner are difficult, if not impossible, to interpret for other reasons as well.
First, all analyses were performed at very high pressure, and were not controlled for hydrostatic pressure effects.
Second, in many instances, no significant differences were observed between the various noble gases, nor between the noble gases and nitrogen.
Third, knowledge of enzyme mode of action and inhibition was very poor at the time of these studies, as were the purities of enzymes used. It is impossible to be certain that confounding enzyme activities were not present or that measurements were made with a degree of resolution sufficient to rank different gases as to effectiveness. Further, any specific mode of action could only be set forth as an untestable hypothesis.
Fourth, solubility differences between the various gases were not controlled, nor considered in the result.
Fifth, all tests were conducted using high pressures of inert gases superimposed upon 1 atmosphere of air, thus providing inadequate control of oxygen tension.
Sixth, all gas effects reported are only inhibitions.
Seventh, not all of the procedures in the work have been fully described, and may not have been experimentally controlled. Further, long delays after initiation of the enzyme reaction precluded following the entire course of reaction, with resultant loss of the highest readable rates of change.
Eighth, the reported data ranges have high variability based upon a small number of observations, thus precluding significance.
Ninth, the levels of inhibition observed are very small even at high pressures.
Tenth, studies reporting a dependence upon enzyme concentration do not report significant usable figures.
Eleventh, all reports of inhibitory potential of inert gases at low pressures, i.e., &lt;2 atm., are postulated based upon extrapolated lines from high pressure measurements, not actual data.
Finally, it is worthy of reiterating that the results of Behnke et al clearly contradict those reported by Schreiner in several crucial respects, mainly that high pressure effects are small and that hydrostatic effects, which were not controlled by Schreiner, are the primary cause of the incorrect conclusions made in those studies.
Additionally, although it was reported by Sandhoff et al, FEBS Letters, vol. 62, no. 3 (March, 1976) that xenon, nitrous oxide and halothane enhance the activity of particulate sialidase, these results are questionable due to the highly impure enzymes used in this study and are probably due to inhibitory oxidases in the particles.
To summarize the above patents and publications and to mention others related thereto, the following is noted.
Behnke et al (1969), disclose that enzyme-catalyzed reactions are influenced by inert gases at high pressures. J. Food Sci. 34: 370-375.
Schreiner et al (1967), describe inert gas interactions and effects on enzymatically, active proteins. Abstract No. 2209. Fed. Proc. 26:650.
Schreiner, H. R. 1964, Technical Report, describes the physiological effects of argon, helium and the rare gases. Contract Nonr 4115 (00), NR: 102-597. Office of Naval Research, Washington, D.C.
Schreiner, H. R. 1965, Technical Report, describes the physiological effects of argon, helium and the rare gases. Contract Nonr 4115 (00), NR: 102-597. Office of Naval Research, Washington, D.C.
Schreiner, H. R. 1966, Technical Report, describes the physiological effects of argon, helium and the rare gases. Contract Nonr 4115 (00), NR: 102-597. Office of Naval Research, Washington, D.C.
Doebblere, G. F. et al, Fed. Proc. Vol. 26, p. 650 (1967) describes the effect of pressure or of reduced oxygen tension upon several different enzymes using the gases Kr, Xe, SF.sub.6, N.sub.2 O, He, Ne, Ar and N.sub.2. All gases were considered equal in their effect.
Colten et al, Undersea Biomed Res. 17 ( 4 ), 297-304 (1990) describes the combined effect of helium and oxygen with high pressure upon the enzyme glutamate decarboxylase. Notably, only the hyperbaric inhibitory effect of both helium and oxygen and the chemical inhibitory effect of oxygen was noted.
Nevertheless, at present, it is known that enzyme activities can be inhibited in several ways. For example, many enzymes can be inhibited by specific poisons that may be structurally related to their normal substrates. Alternatively, many different reagents are known to be specific inactivators of target enzymes. These reagents generally cause chemical modification at the active site of the enzyme to induce loss of catalytic activity, active-site-directed irreversible inactivation or affinity labeling. See Enzymatic Reaction Mechanisms by C. Walsh (W. H. Freeman & Co., 1979). Alternatively, certain multi-enzyme sequences are known to be regulated by particular enzymes known as regulatory or allosteric enzymes. See Bioenergetics, by A. L. Leninget (Benjamin/Cummings Publishing Co., 1973 ).
Traditionally used gas sensors include chemical sensors and semiconductor devices. For example, has been developed a chemical gas sensor by using organically modified silicates as gas-sensing substances. The gas sensor consists of thin layers of organically modified silicates coupled to thin film interdigital capacitors (IDC). Changes in the dielectric properties of the modified silicates, which are caused by gases such as NO.sub.2 NH.sub.3 and SO.sub.2 are reflected by a change in the capacitance and conductivity of the capacitor.
U.S. Pat. Nos. 5,085,760, 5,032,248, 4,988,539, 4,792,752, 4,713,646, and 3,719,564 describe electrochemical gas sensors.
U.S. Pat. No. 5,071,770 describes the development of a nitrous oxide electrode which is based upon a chemically specific interaction.
U.S. Pat. No. 4,227,984 describes a gas sensor using an ion transporting membrane.
SU 631812 describes an electrochemical sensor for oxygen that includes a polymer membrane.
JP 53149395 describes an electrochemical sensor for oxygen.
DE 2808165 describes a potentiostatic solid polymer electrolyte gas sensor.
SU 474727 describes a gas detector using a potentiometric sensor.
DE 2237793 describes a gas sensor to measure blood gases involving the use of a composite membrane, with two types of permeability towards interacting and non-interacting gases.
U.S. Pat. No. 5,060,529 describes a semipermeable membrane probe for invasive detection of gases in a sealed package which is amperometric.
All of the sensors described herein are restricted to the detection of chemicals or elements without the application of enzymes.
A second group of sensors are those which are enzyme-based.
Four categories of biosensors can be distinguished (Danilov and Ismailov, 1989; Graham and Moo-Young, 1985):
1. Electrochemical biosensors (oxygen- and ion-selective electrodes with immobilized enzymic membranes)
2. Immunological and bioaffinity (electrodes containing an enzyme or any other protein or an antibody)
3. Optical (fiber-optic bundles with immobilized enzymes)
4. Bioelectronic (based on semiconductors and biological materials)
Enzymes can be immobilized (Gebelein, 1985). Adsorption (ADEw), covalent bonding (CBE), cross-linking (CLE), matrix entrapment (MEE), and membrane encapsulation (EIM) are the methods used to immobilize enzymes (Treyan, 1980).
Enzyme biosensors are enzyme electrodes, wherein one or several enzymes are coupled to an electrochemical sensor that produces a signal proportional to the quantity of substrate consumed or product formed during the catalytic reaction. The enzyme is usually entrapped in an inert membrane matrix which is physically conjugated to the sensor. The performance of the biosensor (linear response, sensitivity, response time, operational stability) is directly linked to the characteristics of the enzyme membrane (porosity, thickness, stability).
To cite some examples of enzyme electrodes: acetylcholine and urea can be determined respectively by means of an acetylcholine esterase electrode (detection of a decrease in pH) and of an urease electrode (detection of an increase in pH); penicillin G can be determined by using a penicillinase electrode; glucose levels can be measured by a glucose oxidase electrode.
U.S. Pat. No. 4,721,677 describes an enzyme-based sensor for measuring glucose levels, which contains an oxygen reservoir, from which the oxygen is fed to the enzyme through an oxygen permeable membrane.
U.S. Pat. No. 5,120,420 describes the assembly of enzyme/electron acceptor based biosensors.
U.S. Pat. No. 4,885,077 describes a membrane enzyme combination sensor assembly.
A third group of sensors are those which are gas biosensors.
Notably, no enzyme-based sensors for noble gases have been developed.
Development of gas biosensors is based on biological or enzymatic materials. Enzymes are of interest because of their substrate specificity, which confers a higher selectivity to the gas biosensors in comparison to the traditional chemical sensors.
The following enzyme-based gas sensors have been developed.
Notably, several diffusion dependent enzyme badges have been developed to measure toxic gaseous compounds in the workplace atmosphere (hydrogen peroxide, formaldehyde, acetaldehyde, and ethanol). The gaseous substrate molecules reach the badge surface by diffusion, where the enzyme is located, and are converted. The formation of colored compounds due to the combined chemical reagent allows the following of the enzymatic conversion. Color comparison permits to determine the concentration of the gaseous compound.
However no enzyme-based sensors for noble gases have been described. This is because all previous sensors have depended upon chemical reactions of the gas with the sensors, and noble gases are not capable of such reactions.
Thus, a need exists for a means by which gases may be detected.